Process of high purity albumin production

ABSTRACT

A process is provided for the preparation of albumin which has extremely low levels of or is essentially free of colorants, metal ions, human proteins, host proteins, fragments of albumin, polymers or aggregates of albumin and viruses, and which is essentially non-glycated, relatively high in free thiol and with an intact C-terminus. The process comprises passing albumin (preferably expressed and secreted by transformed yeast) through positive mode cation exchange and then positive mode anion exchange chromatography. Other steps may also be employed, for example ultrafiltration, gel permeation chromatography, affinity chromatography binding the albumin (for example using blue dyes) and affinity chromatography binding contaminants (for example using an aminophenylboronic acid resin). Elution of albumin, with a compound having affinity for albumin, from a material having no specific affinity for albumin is also disclosed, as is removal of ammonium ions with a counter-ion.

[0001] This application is a continuation of U.S. application Ser. No.08/952,558, which is a continuation-in-part of U.S. application Ser. No.08/378,859, filed May 25, 1995, the contents of which are herebyincorporated herein by reference.

[0002] The present invention relates to purifying the protein humanserum albumin (HSA) extracted from serum or recombinant human albumin(rHA) produced by transforming a microorganism with a nucleotide codingsequence encoding the amino acid sequence of human serum albumin. Inthis specification, the term Aalbumin≅refers generically to HSA and/orrHA.

[0003] Albumin is used to treat patients with severe burns, shock orblood loss. It is also used to supplement media used for growing highereukaryotic cells and as an excipient in the formulation of therapeuticproteins. At present, the demand for the product is satisfied by albuminextracted from human blood. Examples of extraction and separationtechniques include those disclosed in: JP 03/258 728 on the use of acation exchanger; EP 428 758 on the use of anion exchange followed bycation exchange; and EP 452 753 on the use of heating, adding salt anddiafiltering.

[0004] The production of rHA in microorganisms has been disclosed in EP330 451 and EP 361 991. Purification techniques for rHA have beendisclosed in: WO 92/04367, removal of matrix-derived dye; EP 464 590,removal of yeast-derived colorants; and EP 319 067, alkalineprecipitation and subsequent application of the rHA to a lipophilicphase having specific affinity for albumin.

[0005] The present invention provides highly purified albumin.

[0006] One aspect of the present invention provides a process forpurifying albumin, the process comprising the steps of applying arelatively impure albumin solution to a chromatographic material forwhich the albumin has no specific affinity such that albumin binds tothe material, and eluting the bound albumin from the material byapplying a solution of a compound having a specific affinity foralbumin. Preferably, the chromatographic material is a cation exchanger,such as SP-Sepharose FF, SP-Spherosil etc, as listed below in Example 2.

[0007] The compound with specific affinity for albumin may be octanoate(eg sodium octanoate), other long chain (C₆ to C₂₂) fatty acids,salicylate, octylsuccinate, N-acetyltryptophan or a mixture of two ormore of these.

[0008] A second aspect of the invention provides a process for purifyingalbumin, the process comprising the steps of subjecting an albuminsolution to cation exchange chromatography in which the albumin is boundto a cation exchange material and then anion exchange chromatography inwhich the albumin is bound to an anion exchange material.

[0009] The albumin which is eluted from the cation exchange material maybe subsequently treated by one or more of affinity chromatography,ultrafiltration and gel permeation before being subjected to the saidanion exchange chromatography. Hence, in a preferred embodiment, theprocess comprises the steps of:

[0010] (a) passing an albumin solution through a cation exchange matrixunder conditions such that the albumin will bind to the matrix;

[0011] (b) eluting from said matrix an albumin-containing cationexchange eluate;

[0012] (c) passing said eluate through an affinity matrix comprising analbumin-binding compound;

[0013] (d) eluting from said matrix an albumin-containing affinitymatrix eluate;

[0014] (e) passing said eluate, optionally after ultrafiltration,through a gel permeation matrix to obtain a fraction enriched inalbumin;

[0015] (f) passing the said albumin-enriched fraction through an anionexchange matrix under conditions such that albumin will bind to thematrix; and

[0016] (g) eluting from said anion exchange matrix a purifiedalbumin-containing product.

[0017] Alternatively, the albumin which is eluted from the cationexchange material may be applied to the said anion exchange materialwithout any intervening treatment (other than dilution). Hence, a secondpreferred embodiment provides a process for purifying albumin,comprising the steps of:

[0018] (a) passing an albumin solution through a cation exchange matrixunder conditions such that the albumin will bind to the matrix;

[0019] (b) eluting from the matrix an albumin-containing cation exchangeeluate;

[0020] (c) passing the cation exchange eluate through an anion exchangematrix under conditions such that the albumin will bind to the matrix;

[0021] (d) eluting from the anion exchange matrix an albumin-containinganion exchange eluate;

[0022] (e) passing the anion exchange eluate through an affinity matrixcomprising an albumin-binding compound;

[0023] (f) eluting from the affinity matrix an albumin-containingaffinity matrix eluate;

[0024] (g) passing the affinity matrix eluate through a gel permeationmatrix to obtain a fraction enriched in albumin.

[0025] Preferably, prior to the cation exchange step, the albuminsolution is conditioned by adding octanoate and/or other albuminstabiliser (eg sodium acetyltryptophanate) thereto to a finalconcentration of from about 1-10 mM and adjusting the pH to about4.0-5.0.

[0026] Advantageously, the albumin retained in the cation exchange stepis washed with a high salt solution (eg 0.5-2.0 M NaCl buffered at pH4.0 with 10-100 mM, preferably 20-40 mM, for example 27 mM sodiumacetate) before being eluted.

[0027] Preferably, in processes in which the cation exchange eluate ispassed directly to the anion exchanger, the albumin is eluted in thecation exchange step using a buffer containing a compound having aspecific affinity for albumin, especially an acid or salt thereof, forexample octanoate or any other long chain (C₆-C₂₂) fatty acid,salicylate, octylsuccinate or N-acetyltryptophan.

[0028] Suitably, the albumin is eluted from the anion exchanger with abuffer containing a high level (eg at least 50 mM, preferably 50-200 mM,for example 80-150 mM) of a boric acid salt, for example sodium orpotassium tetraborate.

[0029] The albumin purified in accordance with the invention may then,with or without intervening process steps, be subjected tochromatography on a resin containing an immobilised compound which willselectively bind glycoconjugates and saccharides, such asaminophenylboronic acid (PBA).

[0030] In any process of the invention which involves affinitychromatography, the affinity chromatography preferably uses a resincomprising an immobilised albumin-specific dye, such as a Cibacron Bluetype of dye, preferably immobilised on the resin via a spacer such as1,4-diaminobutane or another spacer of C₁₋₈, preferably C₁₋₆, eg C₁₋₅and most preferably C₄ length, preferably having α,ω-diaminosubstitution. Surprisingly, we have found that such dyes actually have agreater affinity for a 45 kD albumin fragment which can be produced incultures of HA-secreting microorganisms, than they do for the fulllength albumin molecule. The 45 kD fragment typically consists of the1-403 to 1-409 region and is disclosed in Sleep et al (1990)Bio/Technology 8, 42-46 and in WO 95/23857.

[0031] The purified albumin solution prepared by the process of theinvention may be further processed according to its intended utility.For example, it may be ultrafiltered through an ultrafiltration membraneto obtain an ultrafiltration retentate having an albumin concentrationof at least about 80 g albumin per litre, with the ultrafiltrationretentate being diafiltered against at least 5 retentate equivalents ofwater. It can be advantageous to include ammonium ions in certainchromatographic steps, for example in the step involving immobilisedaminophenylboronate. Surprisingly, we have found that such ammonium ionsare relatively tightly bound to the albumin. It is preferable for suchammonium ions to be removed from the albumin and we have found that thiscan be achieved by use of a counter-ion. The desirability of exposingthe albumin to a counter-ion would not have occurred to those in thisart since prior processes have not involved ammonium ions and there wasno reason to suppose that ammonium ions would be bound by the albumin.

[0032] Accordingly, a further aspect of the invention provides a methodof purifying an albumin solution comprising exposing the solution to asolution of a counter-ion such that ammonium ions are displaced from thealbumin and can be removed from the solution.

[0033] The counter-ion (preferably a metal ion such as sodium ions) canbe added to the albumin solution and the ammonium ions removed bydialysis, or the ammonium ion can be diafiltered away across asemi-permeable membrane separating the albumin from the solution of thecounter-ion, or they can be removed by gel permeation chromatography.Diafiltration against at least five retentate volumes of 50 mM sodiumchloride is generally suitable.

[0034] The albumin obtained has been found to have extremely low levelsof, or to be essentially free of, colorants, lactate, citrate, metals,human proteins such as immunoglobulins, pre-kallikrein activator,transferrin, α₁-acid glycoprotein, haemoglobin and blood clottingfactors, prokaryotic proteins, fragments of albumin, albumin aggregatesor polymers, endotoxin, bilirubin, haem, yeast proteins and viruses. By“essentially free” is meant below detectable levels. The term “colorant”as used herein means any compound which colours albumin. For example, apigment is a colorant which arises from the organism, especially yeast,which is used to prepare recombinant albumin, whereas a dye is acolorant which arises from chromatographic steps to purify the albumin.At least 99%, preferably at least 99.9%, by weight of the protein in thealbumin preparations purified by the process of the invention isalbumin. Such highly pure albumin is less likely to cause adverse sideeffects.

[0035] The albumin produced by the process of the invention has beenfound to be at least 99.5% monomeric, preferably substantially 100%monomeric by reducing SDS PAGE, and is characterised by one or more ofthe following characteristics. It has an aluminium ion content of lessthan 150 ng, preferably less than 100 ng; an iron ion content of lessthan 3,000 ng, preferably less than 1,000 ng; a copper ion level of lessthan 10,000 ng, preferably less than 5,000 ng; a magnesium ion level ofless than 3,000 ng, preferably less than 1,500 ng; a zinc ion level ofless than 5,000 ng, preferably less than 3,000 ng, a manganese ion levelof less than 50 ng, all based on one gram of albumin; a glycation levelof less than 0.6, preferably less than 0.15 (more preferably less than0.05), moles hexose/mole protein; a level of low molecular weightcontaminants of below 20 V.sec, preferably less than 10 V.sec, measuredas in Example 9 below; a single peak on a capillary zoneelectrophoretogram; intact, ie homogeneous, C-terminus and N-terminus; afree thiol content of at least 0.85 mole SH/mole protein; and no morethan 0.3 mol/mol of C10 to C20 fatty acids and substantially no C18 orC20 fatty acids.

[0036] The starting material may be an albumin-containing fermentationmedium, or the impure albumin solution may be a solution obtained fromserum by any of the plethora of extraction and purification techniquesdeveloped over the last 50 years, for example those disclosed in Stoltzet al (1991) Pharmaceut. Tech. Int. June 1991, 60-65 and More & Harvey(1991) in ABlood Separation and Plasma Fractionation≅ Ed. Harris,Wiley-Liss, 261-306.

[0037] Especially when the albumin is rHA produced in protease-deficientyeasts or other organisms, the process does not normally comprise a heattreatment step as part of the purification process (in contrast to EP428 758 and EP 658 569). Similarly, if it is prepared frommicroorganisms (rather than from humans) it does not normally require afinal pasteurisation step (typically 60EC for one hour).

[0038] The final product may be formulated to give it added stability.Preferably, the highly pure albumin product of the invention contains atleast 100 g, more preferably 1 kg or 10 kg of albumin, which may besplit between a plurality of vials.

[0039] Although the process of the present invention can be utilised toobtain more purified albumin from an impure albumin solution from anumber of sources, such as serum, it is particularly applicable topurifying recombinant human albumin (rHA). The albumin produced inaccordance with the invention may be any mammalian albumin, such as rat,bovine or ovine albumin, but is preferably human albumin. DNA encodingalbumin may be expressed in a suitable host to produce albumin. Thus,DNA may be used in accordance with known techniques to construct anexpression vector, which is then used to transform an appropriate hostcell for the expression and production of albumin. Such techniquesinclude those disclosed in EP-A-73 646, EP-A-88 632, EP-A-201 239 andEP-A-387 319.

[0040] Many expression systems are known, including bacteria (forexample E. coli and Bacillus subtilis), yeasts (for exampleSaccharomyces cerevisiae, Pichia pastoris and Kluyveromyces lactis),filamentous fungi (for example Aspergillus), plant cells, animal cellsand insect cells. The preferred microorganism is the yeast Saccharomycescerevisiae.

[0041] Exemplary genera of yeast contemplated to be useful in thepractice of the present invention are Pichia (Hansenula), Saccharomyces,Kluyveromyces, Candida, Torulopsis, Torulaspora, Schizosaccharomyces,Citeromyces, Pachysolen, Debaromyces, Metschunikowia, Rhodosporidium,Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis, and the like.Preferred genera are those selected from the group consisting of Pichia(Hansenula), Saccharomyces, Kluyveromyces, Yarrowia and Hansenula.Examples of Saccharomyces spp. are S. cerevisiae, S. italicus and S.rouxii. Examples of Kluyveromyces spp. are K. fragilis and K. lactis.Examples of Pichia (Hansenula) are P. angusta (formerly H. polymorpha),P. anomala, P. pastoris and P. capsulata. Y. lipolytica is an example ofa suitable Yarrowia species.

[0042] It is advantageous to use a yeast strain which is deficient inone or more proteases. Such strains include the well-known pep4-3mutants and strains with mutations in the PRA1 and/or PRB1 genes, as inWoolford et al (1993) J. Biol. Chem. 268, 8990-8998, Cabezón et al(1984) P.N.A.S. 81, 6594-6598, EP-A-327 797 and Jones et al (1982)Genetics 102, 665-677. Alternatively, the proteases in the fermentationmedium may be inactivated by heating. The existence of proteases reducesthe yield of the albumin during the overall process.

[0043] Preferably, the yeast has a low (or zero) level of the Yap3pprotease and/or of the hsp150 heat shock protein, for example as aresult of having the respective genes disrupted, as is taught in ourpatent applications published as WO 95/23857 and WO 95/33833,respectively. Yap3p can cause the formation of the 45 kD albuminfragment referred to below, and hsp150 co-purifies with albumin in someseparation steps.

[0044] Yeast may be transformed with an expression plasmid based on theSaccharomyces cerevisiae 2 μm plasmid. At the time of transforming theyeast, the plasmid contains bacterial replication and selectionsequences, which may be excised, following transformation, by aninternal recombination event in accordance with the teaching of EP 286424. The plasmid may also contain an expression cassette comprising: ayeast promoter (such as the Saccharomyces cerevisiae PRB1 promoter), astaught in EP 431 880; a sequence encoding a secretion leader, forexample one which comprises most of the natural HSA secretion leader,plus a small portion of the S. cerevisiae α-mating factor secretionleader, as taught in WO90/01063; the HSA coding sequence, obtainable byknown methods for isolating cDNA corresponding to human genes, and alsodisclosed in, for example, EP 73 646 and EP 286 424; and a transcriptionterminator, for example the terminator from Saccharomyces ADH1, astaught in EP 60 057.

[0045] The choice of various elements of the plasmid described above isnot thought to be directly relevant to the purity of the albumin productobtained, although the elements may contribute to an improved yield ofproduct.

[0046] Preferred aspects of the invention will now be described by wayof example and with reference to the accompanying drawings, in which:

[0047]FIG. 1 shows schematically a fermenter used to produce rHA;

[0048]FIG. 2 is a UV trace from a C18 PTH Reverse Phase HPLC column(Applied Biosystems Inc), showing the low level of low molecular weightcontaminants in the albumin of the invention;

[0049]FIG. 3 is similar to FIG. 2 but shows low molecular weightcontaminants in prior art albumin;

[0050]FIG. 4 is a gas chromatogram showing the fatty acid content ofcommercially available albumin;

[0051]FIG. 5 corresponds to FIG. 4 but shows albumin of the invention;and

[0052]FIGS. 6a and 6 b show electrospray mass spectrometry for albuminof the invention and prior art albumin, respectively.

EXAMPLE 1

[0053] Preparation of Impure Albumin Solution

[0054] The cloning strategy for construction of the albumin-producingmicroorganism was as disclosed in EP 431 880. Plasmid pAYE316 wasintroduced into a (MATα, leu2, pep4-3, [cir/]) Saccharomyces cerevisiaestrain by the method described by Hinnen et al, (1978) P.N.A.S. 75,1929. Transformants were selected on a minimal medium lacking leucine(Yeast nitrogen base, Difco). When transformants were grown for 72 hoursat 30/C,200 rpm in 5 ml flasks containing either 10 ml of complex (YEP,1% (w/v) yeast extract, 2% (w/v) bactopeptone and 2% (w/v) sucrose), ordefined (0.15% (w/v) yeast nitrogen base without amino acids andammonium sulphate, 0.5% (w/v) ammonium sulphate, 0.1M citricacid/Na₂HPO₄.12H₂O pH6.5, 2% (w/v) sucrose) liquid medium, rHA could bedetected in the cell free culture supernatant by SDS-polyacrylamide gelelectrophoresis and/or by rocket gel immunoelectrophoresis.

[0055] A stock master cell culture in defined liquid medium (BufferedMinimal Medium (BMM) salts medium: Yeast Nitrogen Base [without aminoacids and (NH₄)₂SO₄, Difco], 1.7 g/L; citric acid monohydrate 6.09 g/L;anhydrous Na₂HPO₄, 20.16 g/L, pH 6.5∀0.2, sucrose is added to 20 g/L) isused to prepare running stocks (manufacturer's working cell bank) ofprocess yeast suitable for the preparation of shake flask cultures byfreezing aliquots of the culture in the presence of 20% (w/v) trehalose.

[0056] Fermentation

[0057] This section relates to the production of rHA from stock culturethrough to the final fermentation and is a general definition of an rHAfermentation process which is not limited to the specific detail ofparticular equipment or scale.

[0058] Shake Flask Culture. The yeast [cir^(o), pAYE316] is grown as anaxenic culture physiologically suited for inoculation of the seedvessel. If timing of the seed vessel is to be reproducible, it isnecessary to define the phase of growth (primary carbohydrate excess)and inoculum biomass (12±2 mg/L which requires a 100 ml inoculum per 10litres of medium). One stock vial is inoculated into a shake flaskcontaining 100 mL of BMM+2% (w/v) sucrose and the flask is incubated at30EC on an orbital shaker (200 rpm revolutions per minute) until a celldry weight (cdw) of 0.6-1.2 g/L (assessed by optical density at 600 nm)is obtained. This culture is then used to inoculate a seed fermentationvessel to a level of 12±2 mg/L.

[0059] Seed Fermentation. The inoculum for the main production fermenteris provided by growing the production organism, preferably S. cerevisiae[cir^(o), pAYE316], in a seed fermenter (in this example, 20L workingvolume) to a high cell dry weight (cdw) of approx. 100 gL⁻¹. A fed-batchregime is followed so as to minimise the accumulation of ethanol andacetate and thus to maximise cell yield. The whole of each fermentationis monitored and controlled via a computer control system, such as theMulti-Fermenter Computer System (MFCS) software available from B. Braun(Germany). The software supplied by B. Braun is a Supervisory Controland Data Acquisition Package; similar packages are available from othercompanies. The feed control algorithm is intended to control theaddition of sucrose so that maximum biomass is achieved by avoiding theCrabtree effect, thereby minimising the production of ethanol and/oracetate. The fermentation vessel is subjected to a hot NaOH wash andpyrogen-free water (PFW) rinse. The heat sterilised vessel will containapproximately 10 L of sterile MW10 medium (Table 1) batch salts plustrace elements. The medium for rHA production can be ultrafiltered(10,000 Mol. Wt. cut-off) to remove endotoxins. TABLE 1 MW10 MEDIUMConstituents Batch Medium Feed Medium Salts KH₂PO₄ 2.74 g/L 10.9 g/LMgSO₄.7H₂O 0.58 g/L 2.3 g/L CaCl₂.2H₂O 0.06 g/L 0.24 g/L H₃PO₄ (85% w/w)0.88 ml/L 1.76 ml/L Vitamins Ca pantothenate 20 mg/L 180 mL Nicotinicacid 33.3 mg/L 300 mg/L m-Inositol 20 mg/L 180 mg/L d-biotin 0.133 mg/L0.8 mg/L Thiamine.HCl 16 mg/L 32 mg/L Trace element stock* 10 ml/L 20ml/L Sucrose 0** 500 g/L ZnSO₄.7H₂O 3 g/L FeSO₄.7H₂O 10 g/L MnSO₄.4H₂O3.2 g/L CuSO₄.5H₂O 0.079 g/L H₃BO₃ 1.5 g/L KI 0.2 g/L Na₂MoO₄.2H₂O 0.5g/L CoCl₂.6H₂O 0.56 g/L

[0060] The trace elements are added to demineralised water, acidifiedwith 35 ml/L of 98% H₂SO₄.

[0061] ** 20 g Sucrose/L is added to the batch medium at the 20 L seedfermenter stage. Any convenient method of sterilisation may be used, asmay any depyrogenation method, for example ultrafiltration. The vitaminsare always filter sterilised.

[0062] After the medium is added to the vessel, the operatingtemperature of 30EC is set, as well as the minimum stirrer speed,typically 400-500 rpm. The initial pH is adjusted with ammonia solution(specific gravity 0.901) using a pH controller set at 5.7±0.2. 2M H₂SO₄is also used as a pH corrective agent. Sucrose to 20 gL⁻¹, MW10batchvitamins, and Breox FMT30 antifoam to 0.04 gL⁻¹ are added to the vessel.

[0063] Sterile filtered air is introduced into the vessel at 0.5 v/vim(ie 0.5 litre non-compressed air per litre of medium per minute), themedium is inoculated to 12±2 mg cell dry weight L⁻¹ from an axenic shakeflask culture and the MFCS computer system is initiated. Followingcompletion of the batch phase of growth (signalled by a dissolved oxygentension increase of >15% in 30 min), addition of the feed medium isinitiated, under control of the MFCS system. The control strategy iseffectively the same as described below for the production fermentation.During the fermentation the air flow is increased in two steps in orderto maintain a flow of approximately 1 v/v/m. The dissolved oxygentension (DOT) is controlled at 20% air saturation by changing thestirrer speed. Once the stirrer speed cannot be increased further andthe air flow rate has reached its maximum value, the feed controlalgorithm controls the feed rate to minimise the formation offermentation products. At the end of the feed, the culture istransferred to a production vessel.

[0064] Production Fermentation. An axenic culture of the yeast [cir/,pAYE316] is produced by fed-batch fermentation to a high cdw (>80 gL⁻¹)for the production of extracellular rHA. The production fermenter, inthis example a fermenter with a working volume of 8,000 L, is inoculatedwith the culture grown in the seed fermenter, the cell dry weight ofwhich is preferably >80 g.L⁻¹. The initial cell dry weight concentrationin the production fermenter on transfer of the seed fermenter culture ispreferably 0.25-1.00 g.L⁻¹. Although it is preferred to initiate feedingwithin one hour, it can be delayed if necessary. Due to the very lowvalues of OUR and CER during the initial part of the feed phase and theconsequent errors in their measurement, the automatic control of feedrate using RQ is initially disabled. The feed regime is intended tominimise the accumulation of ethanol and acetate, so as to maximise thecell and product yield.

[0065] The fermentation is carried out in a fermenter such as that shownin FIG. 1, designed to give optimum gas dissolution and bulk mixing. Thevessel, which is subjected to a hot NaOH wash and PFW rinse, willcontain approximately 4000 L of sterile MW10 (Table 1), batch salts andtrace elements. This medium may be sterilised independently of thevessel either by heat or filter sterilisation. It has been found inaccordance with the present invention that it is advantageous for thefermentation medium, such as MW10, to be free of ethylene diaminetetraacetic acid (EDTA), or a salt thereof or other strongmetal-chelating agents, since their presence results in a significantlyhigher degree of coloured contaminants in the albumin produced.

[0066] The operating temperature is set at 30EC, and the stirrer speedregulated to be sufficient to maintain a homogeneous solution, typicallyabout 50 rpm. The initial pH is adjusted with ammonia solution (SG0.901) (controller set to 5.7±0.2). 2M H₂SO₄ may be used as a second pHcorrective agent. The MW10 batch vitamins are added, as is a suitableantifoam, as required (eg Breox FMT30 to 0.125 gL⁻¹).

[0067] Sterile filtered air is added to the vessel at 0.5 v/v/minitially to maximise sensitivity of exhaust gas analysis, and the MFCScomputer system is initiated. The exhaust gas is analysed, for instanceby use of a continuous mass spectrometer (eg a Fisons VG gas analyzer).The vessel is inoculated with the whole of the seed vessel culture(minimum 0.4% v/v). MW10 feed in a volume equal to the batch volume. Thefeed is started and the RQ override control disabled until OUR and CERvalues are sufficiently high to make control effective. The feed rate isadjusted manually during the period without RQ control if RQ isconsistently >1.2. The feed rate is increased, via computer control,according to the following algorithm:

Feed rate (FR)=ke^(μt)

[0068] where k is the initial feed rate, μ is the exponential growthrate, and t is time. The value k is determined empirically as theinitial feed rate that is necessary to achieve a growth rate thatminimises the accumulation of ethanol and acetate. For this example, khas been determined as having a value 0.08 mL of MW10 feed medium perminute per liter of culture. The value μ is related to the maximumgrowth rate of a fully respirative organism, in this example 0.1 h⁻¹.

[0069] t is a counter variable that starts at 0 (zero) and thenincreases by 1 every minute, unless RQ>1.2 or DOT<15%. In these cases,the value of t is reduced.

[0070] The vessel can be overpressured as necessary to enhance OTR. Theculture is held for downstream processing at the end of the feed.

[0071] This hold time should be kept to a minimum, but can be extendedup to 48 hours and beyond if necessary. During the hold phase, thetemperature of the culture is reduced to the minimum possible, typicallybetween 4 and 15/C, preferably 4/C, and the DOT is allowed to fall to0%. The feed is stopped, the aeration turned off and the overpressurereduced. The pH control, however, is maintained. Sufficient agitation ismaintained to retain the cells in suspension and facilitate cooling andpH homogeneity, preferably about 50 rpm.

[0072] The expected yields in accordance with the above procedure are:biomass >80 g cell dry weight/L culture; rHA >1.5 g monomer/L culture(determined by SDS-PAGE, related to the whole culture).

[0073] In order to prepare an impure albumin solution for purificationtreatment in accordance with the present invention when the albumin isrHA, the microorganism cells are removed from the fermentation culturemedium. While it is preferred that the cells be removed prior tobeginning of the purification process as described, it can be carriedout simultaneously with the first step under certain conditions, egwhere the first purification step is carried out in a fluidised bed. Thefermentation culture, which has been cooled in the fermenter during thehold phase to less than 15/C without aeration, is transferred to a tankwhere it is diluted to give a biomass concentration of 180-210 g/kg andcooled further if necessary. The diluted culture should be held for asshort a time as possible without aeration at reduced temperature withsufficient agitation to prevent yeast cell deposition.

[0074] Cells and supernatant are subjected to a primary separation step,for example microfiltration or centrifugation in any appropriatecentrifuge such as an Alfa Laval BTUX510 continuous discharge nozzle runat 5700 rpm. Centrate so produced may be filtered in line, for exampleusing a depth filter (1 μm pore size), supplied by Cuno, to removeresidual whole and broken yeast cells and other particles. At least 75%of the rHA present in the diluted culture is recovered in a single passcentrifugation operation. Optionally, the cell slurry from thisoperation may be resuspended in water or buffer and re-centrifuged toprovide a second centrate, thus enhancing product recovery. Theresultant solution is then treated by the process of the invention topurify the albumin contained therein as shown in Example 2.

EXAMPLE 2

[0075] Purification of Albumin in Accordance with the Invention

[0076] The centrate from a fermentation (such as described in Example1), or an impure albumin solution from any other source (such asplasma), is prepared, or conditioned, for chromatography on a cationexchange matrix while protecting the albumin from polymerisation (byincluding octanoate) and protease activity (by heating or by choosingyeast without damaging levels of proteases). Preferably, sodiumoctanoate is added (Chromatography Solution 13 (CS13)—Table 2) to afinal concentration of 1-10 mM, for example approximately 5 mM, tostabilise the albumin. The pH is adjusted with acetic acid (CS09) to4.3-4.8, preferably 4.50±0.1 (most preferably±0.05), and theconductivity is checked to be >5.5 mS cm⁻¹.

[0077] The culture supernatant from some host strains or speciescontains proteases that can degrade rHA during subsequent processing. Insuch instances, this protease activity can be destroyed by heattreatment of the culture supernatant containing the rHA. Typically 1-10mM sodium octanoate is sufficient to protect the rHA from heatdenaturation, and 30 seconds up to 10 minutes at temperatures of 60-80ECis adequate to inactivate the proteases. Subsequently the supernatantcan be further conditioned as described previously. If degradation byproteases is not encountered, then the heat treatment is preferablyomitted.

[0078] Chromatography

[0079] All operations can be carried out at ambient temperature(20±5/C). The albumin loads (g albumin/L matrix) for the chromatographycolumns are determined from titres of albumin (g/L) by either SDS-PAGE(in the case of the SP-FF column) or GP-HPLC (for all other columns).The progress of each step is monitored by measuring UV absorbance online, for example at 254 or 280 nm.

[0080] The sequence of chromatographic steps as described here is noveland inventive in a number of aspects. The use of a cationic matrix forthe first purification step allows the majority of low molecular weightpigmented species derived from the yeast fermentation to pass directlythrough the column, whereas those that do bind to the matrix are boundweakly and can be removed by a high ionic strength salt clean such as 1MNaCl. Thus the cationic matrix, unlike an anionic matrix which adsorbsthese type of molecules irreversibly, can be regenerated and used formultiple cycles of chromatography as the first step in the purification.Hence, this step forms the basis for a robust commercial chromatographyprocess.

[0081] The use of a Cibacron Blue type of column as the second step inthis example is novel in that it is used specifically to remove a 45 kDafragment of albumin which is very difficult to remove from albumin asits physicochemical properties, eg size and pI, are similar to theintact molecule. Surprisingly, the fragment binds more strongly to thedye than full length albumin does, thus allowing their separation.

[0082] The chromatography solutions used during the purification ofalbumin are detailed in Table 2. Because of the very large scalemanufacture of albumin, and the relatively low cost of the product,these buffer salts are the most suitable for the process as they areavailable in a highly pure form at industrial scale and are low costcompared to other commonly used buffers such as Tris, HEPES or MOPS.Alternative buffers could be used in place of the ones used in Table 2,for example buffers of a similar pK_(a) (eg malate for acetate), but inmost instances cost and availability at large scale rule out their use.Alternative salt forms can be used provided they are soluble, availableat industrial scale and low cost. However, the inclusion of tetraborateions in CS06 and CS10 is particularly advantageous since they perform aspecific role in complexing with carbohydrate moieties in macromoleculesand binding them tightly to the anionic groups on the matrix. Thisresults in an enhanced purity of albumin in the eluate.

[0083] Chromatography can be performed using either axial flow columns,such as those available from Pharmacia, or using radial flow columns,such as those available from Sepragen. In this example, the columns areall axial.

[0084] The buffer solutions can be prepared at the concentrationsdescribed below, or concentrated stock solutions can be prepared andmixed or diluted on-line for immediate use. TABLE 2 CHROMATOGRAPHYSOLUTIONS FOR THE PURIFICATION OF ALBUMIN IN EXAMPLE 2 SolutionConstituent Concentration (gL⁻¹) pH Conductivity (mS cm⁻¹) CS01 SP-FFEquilibrant CH₃COONa.3H₂O 3.69 5.45-5.65 1.9-2.2 CH₃COOH (glacial) 0.220CS02 SP-FF Eluent CH₃COONa.3H₂O 13.6 5.45-5.65 6.5-7.5 CH3COOH (glacial)0.750 CS03 DBA Eluent NaCl 117 9.0-9.4 125-165 CH₃COONH₄ 3.84 NaOH 0.680CS04 0.5 M NaOH NaOH 20.0 >12     80-120 CS05 Gel PermeationCH₃COONa.3H₂O 4.94 5.4-5.6 2.9-3.3 CH₃COOH (glacial) 0.380 Octanoic Acid0.721 NaOH 0.190 CS06 DE-FF Eluent Na₂B₄O₇.10H₂0 7.62 8.9-9.3 11.7-13.5NaCl 5.84 CS07 20 mM NaOH NaOH 0.800 >12    3.5-5.5 CS08 DE-FFEquilibrant CH₃COONa.3H₂O 4.94 5.4-5.6 2.9-3.3 CH₃COOH (glacial) 0.380Octanoic Acid 0.721 NaOH 0.190 CS09 Acetic Acid CH₃COOH Glacial — — CS10DE-FF Wash Na₂B₄O₇.10H₂0 7.62 9.0-9.4 2.3-2.9 CS11 DE-FF Pre-CH₃COONa.3H₂O 61.8 5.5-5.7 24-28 equilibrant CH₃COOH (glacial) 2.98 CS12DBA Equilibrant/ NaCl 11.7 8.8-9.2 18-22 Wash CH₃COONH₄ 0.960 NaOH 0.150CS13 2 M Sodium Octanoic Acid NaOH 288 7.7-8.2 — Octanoate 76.0 CS141.73 M H₃PO_(4 (85%) 200  <1.2 — Phosphoric acid (w/w)) CS15 2 M AmmoniaNH₄OH (30% NH₃ 113 ml — — (w/w))

[0085] Cation Exchange Chromatography. Albumin is concentrated andpurified with respect to at least yeast proteins (if the albumin is rHAfrom a yeast fermentation) and other antigens, low molecular weightcontaminants and pigmented compounds by cation exchange chromatography.The method uses a commercial cation exchange matrix such as SP-SepharoseFF, SP-Spherosil, CM-Sepharose FF, CM-Cellulose, SE-Cellulose orS-Spherodex. Preferably the matrix is SP-Sepharose FF (Pharmacia) at abed height of 5 to 25 cm, preferably 10 to 15 cm and in this example12.5 cm, with a column loading of 10 to 50 g albumin/L, preferably 40±10g albumin/L matrix. The matrix is equilibrated with a buffer to removethe alkali storage solution; preferably the buffer should be strongenough to reduce the pH to approximately pH6.0. A buffer such as CS01 isused to remove storage solution CS07 from the column; however, anybuffer with a pH <6.0 could be used. Equilibration is judged to becomplete when the pH of the column effluent is approximately pH6.0.

[0086] The conditioned centrate is then loaded onto the column at a flowrate of, for example 1.0-8.0 cm/min, preferably 4.0-7.0 cm/min, in thisexample, 6.36 cm/min, and then the column is washed with a solution toremove residual contaminants. This wash solution should have a pH<6.0and a conductivity less than 5 mS cm⁻¹, preferably less than 3 mS cm⁻¹,to prevent the elution of albumin. A suitable solution is CS01. Thepreceding steps are all run at 6.36 cm/min; for elution and allsubsequent steps the flow rate is reduced to 0.5-5.0 cm/min, preferably2.0-4.0 cm/min, in this example 3.18 cm/min, in order to reduce thevolume of eluate. Elution of albumin is effected by increasing the ionicstrength; a solution with a conductivity in the range 5-10 mS cm⁻¹,preferably 6-8 mS cm⁻¹, for example CS02, is used. The collection ofalbumin starts when the UV signal rises above 1.0 A₂₈₀/cm, andcollection continues until the UV signal falls below 0.6 A₂₈₀/cm or amaximal volume of 6.5 column volumes has been collected. The column isthen cleaned using CS03 and 04, and then stored in CS07.

[0087] Affinity Chromatography. This step further purifies the albuminwith respect to a 45 kDa N-terminal albumin fragment, yeast antigens (ifthe albumin is rHA from a yeast fermentation) and pigment. The affinitymatrix may comprise any Cibacron Blue type of dye which binds albumin,for example Reactive Blue 2, Procion Blue HB, Blue Sepharose, BlueTrisacryl and other anthraquinone-type compounds. Preferably, the matrixis the ADelta Blue Agarose≅matrix described below. This has been foundto reduce the levels of Blue leachates generated by the matrix and toenhance the alkaline stability of the matrix to facilitate cleaning anddepyrogenation. A further improvement of the matrix compared tocommercially available matrices is the incorporation of a spacer,1,4-diaminobutane, between the dye (Reactive Blue 2) and the matrix.This was found to be the optimal length of spacer with respect to eluatealbumin purity.

[0088] Reactive Blue 2 has the chemical structure represented below.

[0089] The ortho, meta or para isomer, or any mixture thereof, can beused. The preferred isomer the ortho-SO³⁻ form but, as it is difficultto make to the desired purity, the meta isomer is used. Theaminobutyl-Reactive Blue 2 is prepared to a minimum purity of 98% totalpeak area as determined by analytical HPLC. This can be achieved eitherby using crude commercially available dye, which will necessitatepurification of the aminobutyl derivative dye, or using a puresynthesised dye. In the latter method, the starting dye material shouldbe a minimum of 98% pure by analytical HPLC at 280 nm. Such material isavailable from ACL, Isle of Man. Reactive Blue 2 is reacted with1,4-diaminobutane in water by heating the mixture to 60EC, after whichthe derivatised dye is purified from the mixture, for instance byprecipitation. The aminobutyl-Reactive Blue 2 is then coupled to thematrix, for instance to epichlorhydrin-activated Sepharose CL-6B(Pharmacia, Sweden). See Porath et al (1971) J. Chromatog. 60, 167-177.The dye content of such a Delta Blue Agarose (DBA) matrix should,preferably, be 50 ∀ 5 mmole/g dry weight.

[0090] Use of Blue Matrix. The method uses DBA at a bed height of 10-30cm, preferably 20-30 cm (in this example 25 cm ), with a column loadingof 7-14 g rHA/1 matrix, preferably 8-12 g/l (in this example 10∀ 1 galbumin/L matrix); all steps are run at a flow rate of 0.3-2.0 cm /min,preferably 1.0-2.0 cm /min, in this example 1.53 cm /min. The DBA isequilibrated in CS01 from CS07; equilibration is complete when the pH ofthe column effluent is approximately pH9.5. Prior to chromatography, theSP-FF eluate is adjusted to approximately pH8.5-9.5, preferably pH 9.0,with ammonia, and then loaded onto the column. When loading is complete,the column is washed to remove contaminants with 1-5 volumes of buffer10-30 mS cm⁻¹, preferably 15-25 mS cm⁻¹, for example CS12, preferably 5column volumes. The albumin is eluted using a high ionic strength bufferof<100 mS cm⁻¹, preferably 125-165 mS cm⁻¹, for example CS03. Eluatecollection is started when the UV signal (A₂₈₀/cm) rises above 0.4, andstops when the signal falls below 0.4 again. The column is then cleanedusing CS04 and stored in CS07.

[0091] Intermediate Ultrafiltration. This step concentrates the albuminfor gel permeation chromatography. A cellulose-type membrane (nominalmolecular weight cut off less than or equivalent to 30,000, for example10,000) in an ultrafiltration apparatus is used to concentrate DBAeluate to a retentate concentration of 20-120 g/L albumin, preferably80-110 g /L. The membranes are treated, post-use, by flushing outresidual protein with water, or CS03 or CS05 from Table 3, and cleaningwith 0.1M sodium hydroxide. The membranes may then be stored in 20 mMsodium hydroxide.

[0092] Gel Permeation Chromatography. This step purifies the albuminwith respect to yeast antigens (if the albumin is rHA from a yeastfermentation), pigment and dimerised albumin and performs a bufferexchange step. The method uses a commercial gel permeation matrix suchas Sephadex G100, G150, G250, Sephacryl S-100, S-200 or S-300, ToyopearlHW50S or Superose 6 or 12. Preferably, the matrix is Sephacryl S-200 HR(Pharmacia) at a bed height of greater than 60 cm, preferably 90 ∀cm(3×30 cm ). The column is equilibrated in CS05 and run at 0.1-1.5 cm/min, preferably 0.5-1.0 cm/min, in this example 0.75 cm /min; thecolumn is then loaded with albumin from the intermediate UF step when pH9.5 is reached. The load volume is equivalent to approximately 2-9% ofthe column volume, preferably 5-8%, for example 7.5% of the columnvolume. The albumin fraction is collected in three parts: an initialsmall amount of albumin dimer goes to waste until the A₂₈₀/cm reaches10% full scale deflection (FSD) on the way up; at this point collectionof a recycle fraction starts and continues until 90% FSD and then thealbumin is collected as the primary product fraction. This continuesuntil the A₂₈₀ falls through 5% FSD, after which the effluent stream isdirected to waste again. The recycle and primary product fractions arecollected separately. This step is repeated until all the material hasbeen loaded onto the column.

[0093] S-200 HR Recycle Ultrafiltration. A cellulosic type membrane,nominal molecular weight cut-off equal to or less than 30,000, or asused in this example 10,000, in an ultrafiltration apparatus, is used toconcentrate the pooled recycle fraction to a retentate concentration of20-120 g/L album in, preferably 80-110 g/L. The membranes are treated,post-use as described above under Intermediate Ultrafiltration.

[0094] Alternatively, as in any ultrafiltration steps in this process,polyethersulfone or PVDF membranes with a cut-off of # 30,000 may beused instead of the cellulose-type membranes. Such membranes areavailable from Amicon and Millipore. It is preferable to use membraneswhich are compatible with NaOH, used for storage and cleansing of themembranes.

[0095] Purification of S-200 HR Recycle Ultrafiltration Retentate. Theretentate from recycle ultrafiltration is loaded onto the same column asused for the primary S-200 purification and a product fraction collectedfrom each peak, which is then mixed with the bulked primary productfractions collected previously. This step is repeated until all thematerial has been loaded onto the column.

[0096] Anion Exchange Chromatography. The aim of this step is to purifyalbumin with respect to at least yeast antigens (if the albumin is rHAfrom a yeast fermentation) and pigmented albumin. The method uses ananion exchange matrix such as QMA-Spherosil, DEAE-Spherodex, Q-Hyper D,DEAE-cellulose, QAE-cellulose, or TMAE, DMAE, or DEAE Fractogel.Preferably, the matrix is the commercial anion exchange matrix DEAESepharose-FF (Pharmacia) at any convenient bed height in the range 5-25cm, preferably 10-15 cm, for example 12.5 cm, with a column loading of10-60 g albumin per litre of matrix, preferably 35 ∀ 15 g/L matrix. Thecolumn is first equilibrated in a strong buffer to bring the pH down tothe working range quickly, eg sodium acetate pH 4.5-6.0, preferablyapproximately pH5.5, for example CS11. After the concentrated buffer, asolution of lower conductivity, namely in the range 1-4 mS cm⁻¹,preferably 2.5-3.5 mS cm⁻¹, for example CS08, is used to equilibrate thecolumn prior to loading the column with S200 eluate. A linear flow rateof 1.0-8.0 cm/min, preferably 3.0-7.0 cm/min, in this example 4.4 cmmin⁻¹, can be used. When loading is complete, the column is washed witha solution of sodium tetraborate in the range 5-30 mM, preferably 15-25mM, for example CS10. This causes any carbohydrate-containingcontaminants to adhere to the column more strongly prior to elution ofthe album in fraction. Elution can be effected by any high ionicstrength solution in the range 10-20 mScm⁻¹, preferably with CS06. Theeluate is collected when the A₂₈₀/cm reaches 0.4, and continues untilthe peak falls through 0.8.

[0097] Hence, in this example, the sequence of purification steps is:cation exchange, affinity chromatography, ultrafiltration, gelpermeation (with ultrafiltration of recycle fraction) and anionexchange.

[0098] The eluate from the DE-FF column has been found to have less than0.1% (w/w) albumin dimer and an undetectable level of albumin polymersor aggregates as analysed by GP HPLC using a TSK SW3000XL column, loadedwith 25.0 μl of eluate containing 10.0 mg/ml of albumin.

EXAMPLE 3

[0099] Formulation of Purified Albumin into a Final Product

[0100] This Example illustrates the concentration, diafiltration andformulation of the highly purified albumin into a suitable product, inthis instance 25% (w/v) albumin. This procedure is carried out in twostages, namely final ultrafiltration (UF) and formulation. Final UFbegins with transfer of the DEAE eluate (adjusted to pH 7.0±0.3 withphosphoric acid) to the Final UF feed vessel and terminates afterretentate and washings, if any, are transferred to the formulationvessel. The albumin-containing process stream is sequentially subjectedto primary concentration, diafiltration and secondary concentration inan ultrafiltration system fitted with cellulosic or, more preferably,polyethersulphone membranes with a nominal molecular weight cut offlimit of 10,000. The initial concentration step increases the albuminconcentration to approximately 100 g.L⁻¹ and is immediately followed bythe continuous diafiltration phase where the albumin is diafilteredagainst at least 5, preferably at least 7, retentate volume equivalentsof water-for-injection.

[0101] In some purification processes of the invention, for example thestep set out in Example 7 using immobilised aminophenylboronate,ammonium ions may be present at this stage. Surprisingly, we have foundthat these ammonium ions are bound quite tightly by the albumin andcannot be completely removed by diafiltration against water. We havefound that diafiltration against a salt solution is effective. A ratioof 0.5 to 10% w/w of sodium chloride to albumin, for example 1.0 to 5.0%or about 3%, may be used. The salt may be added to the albumin retentateor, more usually, will be added to the diafiltration water. For anultimate 5% (w/v) formulation, a solution of approx 100 g/l may berecovered directly from the diafiltration step. For an ultimate 25%(w/v) formulation, a solution of approx 275-325 g/l is obtainedfollowing a further concentration step (UF). Finally, the solution istransferred to the bulk product formulation vessel.

[0102] The formulation step produces albumin in an appropriate chemicalenvironment and at an appropriate concentration suitable for bulkproduct sterile filtration (0.22 μm hydrophilicpolyvinylidene-difluoride) and filling. The transferred solution isanalysed to determine concentrations of albumin, sodium and octanoate.These quantities are taken into account and any necessary furtheramounts of stock sodium chloride and sodium octanoate excipientsolutions and appropriate grade water added to achieve the bulkformulation specification. The final albumin concentration may be235-265 g.L⁻¹ (ie about 25%), with a sodium concentration of 130-160 mM.Any other feasible albumin concentration may be made, however, with, forexample, a minimum concentration of at least 4% (w/v), preferably 4-25%(w/v). Formulation is complete following addition of appropriateconventional pharmaceutically acceptable excipients, such as thosespecified in the US or European Pharmacopoeias for human albumin, anddiluting water.

[0103] A final concentration of 0.08 mmoles sodium octanoate per gram ofalbumin may be desirable. The product is sterile and non-pyrogenic.There may be about 1% (w/w) dimeric albumin but no larger polymers oraggregates are detectable as analysed by GP HPLC using a TSK SW3000XLcolumn.

EXAMPLE 4

[0104] Cation Exchange Followed Directly by Anion Exchange

[0105] In a variation of the process of Example 2, the order of thesteps was altered and some changes were made in the process conditions.A further table of chromatographic solutions is therefore provided, asTable 3. In addition, all of the chromatographic columns except the gelpermeation step are radial flow. TABLE 3 CHROMATOGRAPHY SOLUTIONS FOREXAMPLE 4 Solution No. Name Constituent Concentration (g.L⁻¹⁾ pH(mS.cm⁻¹) CS20 SP-FF Equilibrant/Wash/ CH₃COOH 1.85 5.45-5.65 1.9-2.2DE-FF Equilibrant NaOH (27% (w/w)) 4.00 CS23 SP-FF Eluent/ CH₃COOH 5.135.4-5.6 5.0-6.0 DE-FF Pre-Equilibrant NaOH (27% (w/w)) 11.5 OctanoicAcid 0.721 CS24 SP-FF/DE-FF Salt Clean NaCl 58.4 5-9 75-95 Tween 80 5.00CS25 0.5 M NaOH (UF membrane NaOH (27% (w/w)) 74.1 >12  80-120 clean)CS26 20 mM NaOH NaOH (27% (w/w)) 2.96 >12 3.5-5.5 CS27 DE-FF WashK₂B₄O₇.4H₂O 4.80 9.0-9.4 2.5-3.5 CS29 DBA Equilibrant/Wash CH₃COONH₄19.3 NaOH (27% (w/w)) 593 8.7-9.1 18-22 CS30 DBA Eluent NaCl 117 6.7-7.1125-165 NaOH (27% (w/w)) 14.1 H₃PO₄ (85% (w/w)) 5.79 CS32 0.1 M NaOH (UFmembrane NaOH (27% (w/w)) 14.8 >12 16-24 storage) CS33 2 M SodiumOctanoate NaOH (27% (w/w)) 281 7.8-8.4 — Octanoic Acid 288 CS34 AceticAcid CH₃COOH 1045 — — CS35 0.5 M Phosphoric Acid H₃PO₄ (85% (w/w)) 59.0 <1 —

[0106] The initial cation exchanger step was essentially the same as inExample 2, but with the following variations. The bed flow path lengthwas 11.0 ∀ 1.0 cm . The chromatography was then carried out as follows.

[0107] An SP-FF (Pharmacia) column was equilibrated in four volumes of10-100 mM acetate, preferably 20-40 mM, for example 30 mM as in CS20,and the albumin solution was loaded at a flow rate of 0.07 to 0.75 bedvolumes per min, preferably 0.3-0.6, in this example 0.5 bed volumes perminute. The column was washed with eight volumes of 10-100 mM,preferably 30-70, for example 50 mM acetate (CS21) and then ten volumesof CS20 and the albumin eluted with, and collected in, anacetate/octanoate buffer (for example 40-120, preferably 60-100, eg 85mM acetate, and 2-50, preferably 2-20, eg 5 mM octanoate, as in CS23)using an A₂₅₄/cm of 0.6 and 0.36 to mark the start and end ofcollection. The column is cleaned with 0.25-3.0 M salt and 0.05-2%detergent (CS24) and then 0.1-1.0 M caustic (CS25) and stored in dilute(10-50 mM) caustic (CS26). In this example, the flow rate for theequilibration, loading and washing steps is 0.5 bed volumes per minute.For elution of the albumin, a flow rate of 0.04-0.6 bed vol/min,preferably 0.15-0.35, in this example 0.25 bed vol/min is used. Theanticipated recovery of rHA monomer is between 46 and 66%.

[0108] The albumin was therefore eluted from the cation exchange columnwith a solution of octanoate, achieving a novel biospecific elution ofrHA from a cation exchanger. The pH is close to the pI of the albumin sothat the binding of the octanoate causes a significant overall chargedifference; for example, the pH is at least 4.5, preferably about pH5.5.

[0109] The eluate from the cation exchanger is then loaded directly (ieinstead of after affinity and gel permeation chromatography as inExample 2, but preferably after dilution) onto the anion exchange resinat a pH of 4.5-6.5, preferably about 5.5, and a conductivity preferablyin the range 1.5 to 5.0 mS.cm⁻¹, for example 2.5 ∀ 0.5 mS.cm⁻¹. This hasbeen found to result in any dimeric albumin that was formed during thecation exchange chromatography being converted back to monomeric albuminunder the conditions of the anion exchange chromatography. A yield ofapproximately 110% for albumin monomer has been achieved over this step.

[0110] In more detail, an 11.0±1.0 cm bed flow path length column ofDEAE-Sepharose Fast Flow (Pharmacia) is pre-equilibrated with the cationexchange elution buffer (CS23) and then equilibrated with an acetatebuffer (for example CS20) before being loaded with 30.0 ∀ 10.0 gmonomeric albumin per litre of matrix.

[0111] The column is then washed with a borate solution as in Example 2(CS27), eluted as in Example 2 (CS06), and cleaned with salt/detergent(CS24), caustic (CS25) and stored in dilute caustic (CS26) all as forthe cation exchange column. The flow rate for all the steps is 0.07 to0.75 bed vol/min, preferably 0.3-0.6, in this example 0.5 bed volumesper minute.

[0112] The eluate from the anion exchange resin (eg DE-FF) stillcontains impurities and is then applied directly to the affinity matrix(eg Delta Blue Agarose as described in Example 2). The bed height wasreduced from 25 cm in Example 2 to 11.0±1.0 cm which allowed a higherflow rate within normal operating pressure. Therefore, a bed height of11.0 cm was preferred and does not adversely affect recovery of albuminor albumin purity. The column was equilibrated in ammonium acetate(100-300 mM, preferably 200-275, for example 250 mM as in CS29) and thealbumin was applied at 7.0-14.0 g/l, preferably 8.0-12.0 g/l, in thisexample 10.0±1.0 g per litre of matrix. Equilibration, load and washsteps were performed at flow rates of 0.05-0.30 bed vol/min, preferably0.15-0.27, in this example 0.25 bed vol/min. All other steps wereperformed at 0.04-0.30, preferably 0.1-0.25, and in this example, 0.20bed vol/min. The increased flow-rate, facilitated by the reduced bedheight, improved the throughput by a factor of four which isadvantageous to the large scale plant design and was close to themaximum operating capability of the DBA. Since this increased flow ratedid not appear to adversely affect recovery of albumin or albuminpurity, it is preferred to utilise such a higher flow rate.

[0113] The column was washed with 5 column volumes of the ammoniumacetate buffer (CS29), and the albumin was eluted with strong salt andphosphate solution (1.0-3.0 M NaCl, for example 1.5-2.5 M or 2.0 M NaCl,and 5-100 mM, eg 50 mM phosphate, as in CS30).

[0114] The pH of the eluant in this variant of the process was changedto pH7.0 from pH9.2. The buffer was changed accordingly from 50 mMammonium acetate to 50 mM sodium phosphate which was preferred becauseof its buffering at pH7.0, and its relative cost. The lower pH eluantwas responsible for an increase in DBA eluate albumin monomer recovery.A pH lower than 7.0 increased the fragment levels, and above pH7.0 thealbumin monomer recovery was reduced. The pH, which can be in the range5.5-9.0, is therefore preferably pH7.0. The column was cleaned andstored in caustic (CS25, CS26) as above.

[0115] The DBA eluate (optionally after ultrafiltration with acellulosic type membrane (nominal cut off MW30,000) to give 80-110 g/lof albumin) was then applied to the gel permeation resin, for exampleS-200 (HR). The S-200 running buffer was changed to 40 mM sodiumphosphate pH7.0. The sodium octanoate was omitted from this buffer forcost reasons, and instead was added to the solution prior todiafiltration (added to a concentration of 1-20 mM, preferably 5 mM).The phosphate conferred a higher conductivity on the running bufferwhich improved the purity. A high salt concentration can be used toincrease conductivity but it is still preferable to buffer the solution.The pH7.0 was preferable since this was the desired pH for formulation.

[0116] Hence, in this example, the sequence of purification steps is:cation exchange (eluting with a molecule specifically bound by albumin),anion exchange, affinity chromatography and gel permeation.

[0117] The diafiltration step prior to formulation may be assisted bystarting with albumin at pH7.0. The albumin was more concentrated at thefinal eluate than with the process of Example 2, assisting the finalultrafiltration step prior to formulation (Example 3).

EXAMPLE 5

[0118] High Salt Wash on Cation Exchanger

[0119] In a further variation of the process, the process of Example 2or 4 was followed except as follows. Following loading of the albumin onto the cation exchange column (for example SP-Sepharose FF, Pharmacia),the column was washed with CS21 (50 mM sodium acetate, pH 3.9-4.1,0.6-0.8 mS.cm⁻¹), then further washed with a high salt buffer containing1-3M NaCl, preferably 2M NaCl, in sodium acetate buffer (for example10-50 mM sodium acetate, preferably about 27 mM, pH 3.5-4.5, preferablypH4.0) before the final wash in CS20. This more stringent washingprocedure results in an eluate containing a lower level of non-albuminproteins and may be especially useful if the albumin is rHA from a yeastfermentation. The albumin was eluted as described in Example 4. Thelowering of the pH prior to the high salt wash helps to retain thealbumin on the column during that wash, and the final wash alsomaximises albumin recovery. It is probable that neither step has a majoreffect on the purity of the albumin recovered.

EXAMPLE 6

[0120] Concentrated Borate Elution from Anion Exchanger

[0121] In this example, the process of Example 2 or 4 (with or withoutthe variation in Example 5) was varied as follows. The eluate from thecation exchange column was diluted to below 10 mS.cm⁻¹, preferably lessthan 5 mS.cm⁻¹, and then loaded on to an anion exchange matrix (forexample DEAE Sepharose FF, Pharmacia). The anion exchange matrix wasthen washed with dilute tetraborate buffer (for example 15-25 mMpotassium tetraborate or sodium tetraborate), which has the effect ofraising the pH to approximately 9.2, and then the albumin was elutedwith a more concentrated tetraborate buffer (for example 80-150 mMpotassium tetraborate, preferably 110 mM potassium tetraborate). InExamples 2 and 4, the albumin was eluted with 20 mM tetraborate, 100 mMNaCl; elution with 80-150 mM tetraborate (eg 33.6 g/l) results in aneluate with a lower content of carbohydrate-containing contaminants, forexample yeast glycoproteins, due to an increased affinity of thesespecies for the anion exchange matrix under these conditions. Potassiumtetraborate is used in preference to sodium tetraborate because of itshigher solubility at room temperature. The eluate from the anionexchange matrix was dealt with as in Example 2 or 4. For example, in theExample 4 process, it was then directly loaded onto an affinity matrix,eg Delta Blue Agarose (DBA), which was run as described in Example 4.

[0122] A gel permeation step is then carried out as in Example 2 or 4.

EXAMPLE 7

[0123] Immobilised Aminophenylboronate

[0124] The eluate from the DBA matrix may be applied to a gel permeationmedium, for example Sephacryl S-200 (HR) (Pharmacia), equilibrated in anammonium acetate buffer (for example 10-100 mM, preferably about 30 mM),containing sodium chloride (20-2000 mM, preferably about 100 mM) andoctanoate (1-20 mM, preferably about 5 mM octanoate at pH 9.0-9.5,preferably 9.2). This buffer effectively exchanges the albumin into asuitable solution for the final chromatographic step, set out in moredetail below.

[0125] The S-200 step is run as follows. The S-200 is run at a minimumbed height of 90.0±3 cm (eg 3×30 cm in series). (a) The retentate fromintermediate ultrafiltration is loaded onto the column. Recycle andproduct fractions are collected. This step is repeated until all thematerial has been loaded onto the column. (b) The pooled recyclefractions are concentrated to 80-110 g rHA/L by ultrafiltration asabove. (c) The retentate from recycle ultrafiltration is loaded onto thesame column and a product fraction collected from each peak. This stepis repeated until all the material has been loaded onto the column. (d)The product fractions from the primary and secondary gel permeationchromatography steps ((a) and (c)) are pooled as the S-200 eluate.

[0126] The final step consists of an affinity step to removeglycoconjugates, such as glycoproteins and glycolipids, and poly-,oligo- and monosaccharides. This step uses immobilisedaminophenylboronic acid (PBA) as the ligand. U.S. Pat. No. 4 562 251(incorporated herein by reference) describes suitable methods for makingdiborotriazine agarose or monoborotriazine agarose: (1) Triazine isO-linked to agarose first and then linked with 3-aminophenylboronic acid(APBA) in a second reaction. If the X on the triazine is replaced withchlorine then the disubstituted resin is produced. (2) Triazine isreacted with APBA first to produce either mono or diborotriazine. Theseare then O-linked via the free chlorine on the triazine to the —ONaactivated agarose to produce either mono or disubstituted agarose. Allof the examples and descriptions in this patent use —ONa activatedagarose which results in O-linkages.

[0127] An earlier patent U.S. Pat. No. 4,269,605 contemplates a varietyof matrix activation methods, including epichlorohydrin activation ofagarose, preferred herein. Commercially available matrices includeAmicon's PBA30 and Sigma's acrylic beaded aminophenylboronate.

[0128] The albumin collected from the S-200 column was chromatographedthrough the PBA matrix, having been pre-equilibrated in S-200 runningbuffer (see above); under these conditions, the albumin does not bindappreciably to the matrix, whereas the carbohydrate-based contaminantsare retarded sufficiently to separate them from the albumin as it passesthrough the column. The chromatography is thus in the negative mode withrespect to the albumin. Further details were as follows:

[0129] The phenyl boronate matrix had a flow path length of 11.0±1.0 cmand was equilibrated with a buffer containing ammonium ions (10-50 mM),acetate (10-50 mM) and 1.0-10.0 mM octanoate (eg CS36—see table below).The column was then loaded at 35±15 g of rHA/L matrix. The PBA is run asa negative step and therefore the product collected is the flow throughduring loading and the subsequent wash with the equilibration buffer.All chromatographic steps can be performed at flow rates in the range0.005-0.3 bed vol/min. Preferably equilibration and cleaning of thecolumn are carried out at a higher flow rate, eg 0.19 bed vol/min, thanload and collection of the albumin solution, which is preferably carriedout at a flow rate of 0.01-0.05, preferably 0.025 bed vol/min. Thecolumn is then cleaned with a borate buffer (as in CS37), salt (CS38)and caustic (CS25) and then stored in the borate buffer (CS37).

[0130] The pH of the collected flow through and wash is adjusted to7.0±0.1 with phosphoric acid solution (CS35).

[0131] The buffers used are as follows: TABLE 4 Chromatography solutionsfor Example 7 Solution Concn Conductivity No. Name Constituent (g/l) pH(mS.cm⁻¹) CS36 PBA CH₃COONH₄ 2.31 9.0-9.4 12.0-15.0 equil- NaOH 2.55ibration/ (27% w/w) wash NaCl 5.84 Octanoic acid 0.721 CS37 BorateK₂B₄O₇.4H₂O 33.6 9.2-9.5 15.0-18.0 clean CS38 Salt clean CH₃COOH 1.623.9-4.1 125.0-165.0 NaOH 1.19 (27% w/w) NaCl 117.0

[0132] Because of the use of ammonium ions in the PBA buffer, it isadvantageous to use salt in the final ultrafiltration step, as explainedin Example 3 above.

[0133] In a particularly preferred process, the sequence of steps is asfollows:

[0134] (1) Yeast fermentation as in Example 1.

[0135] (2) Centrate conditioning as in Example 2.

[0136] (3) Cation exchange (SP-FF) with high salt wash, as in Example 5,and elution with albumin-specific compound, as in Example 4.

[0137] (4) Dilution and anion exchange with concentrated tetraborateelution as in Example 6.

[0138] (5) Affinity chromatography (DBA) as in Example 4.

[0139] (6) Intermediate ultrafiltration and then gel permeation (S-200),with recycle ultrafiltration, as in Example 7.

[0140] (7) Chromatography on immobilised borate as in Example 7.

[0141] (8) Final ultrafiltration and formulation as in Example 3.

EXAMPLE 8

[0142] Earlier use of Immobilised Phenylboronate

[0143] The step involving immobilised phenylboronate may be used earlierin the process, for instance in a process in which the steps areordered: cation exchanger—anion exchanger—affinitymaterial—ultrafiltration/diafiltration—immobilised phenylboronate—gelpermeation.

[0144] The conditions for each step are as in Examples 4 to 7, except asfollows. The DBA eluate is concentrated to 80-110 g/l albumin and the pHis adjusted to 9.2 by diafiltering (5 volumes) against an ammoniumacetate of the kind used in Example 7. The concentrated DBA eluate isthen chromatographed on PBA and the flowthrough is collected and applieddirectly to the gel permeation (eg S200) column. As the gel permeationstep is now the last step, it may run in a buffer which is suited to theformulation step, for example 20-130 mM (preferably 50-100 mM) NaCl, atpH 7.0.

EXAMPLE 9

[0145] Characterisation of the Albumin Produced According to theInvention

[0146] This Example illustrates the analysis that is carried out toestablish the purity of albumin purified in accordance with the presentinvention. Unless stated otherwise, all of the assays are performed onalbumin which has been formulated as described in Example 3 to yield thefinal product.

[0147] Glycation of rHA

[0148] A microassay for glycated protein has shown that (rHA) purifiedin accordance with the invention is not modified by non-enzymicglycosylation (glycation). The microassay measures the stable Amadoriproduct (AP) form of glycated protein, by oxidation of the C-1 hydroxylgroups of AP with periodate. The formaldehyde released by periodateoxidation is quantitated by conversion to a chromophore,diacetyldihydrolutidine (DDL), by reaction with acetylacetone inammonia. DDL is then detected colorimetrically at 405 nm. Albumin batchMole hexose/mole protein A 0.092 B 0.116 C 0.090 D 0.132 E 0.060 G 0.04H 0.01 I 0.07 J 0.07 K 0.05 L 0.740 M 0.70 N 0.96 O 0.78

[0149] Batches A-K were rHA purified according to Example 2. Batches L-Owere samples of commercially available human serum albumin fromdiffering sources. Eight batches of rHA purified according to Example 7had a negligible level of glycation (0.042±0.018 moles/mole) compared toHSA (0.387±0.012).

[0150] Low Molecular Weight Contaminant Assay

[0151] Rationale—The aim of this assay is to remove non-covalently boundlow molecular weight contaminants (LMC) from rHA and HSA using acidicorganic solvents. An HPLC “fingerprint” chromatogram can then beproduced for comparison of samples.

[0152] Method—To 100 μl of final product (20 mg; rHA or HSA) is addedsequentially 50 μl formic acid (98% v/v), 100 μl chloroform and 50 μlethanol with vortexing after each addition. The samples are kept at roomtemperature for 5 mins with regular mixing. Protein is then precipitatedby the addition of 1 ml acetone (30 mins, −20EC). The protein samplesare pelleted by centrifugation and the supernatants are decanted off anddried by rotary evaporation under vacuum. The dried samples areresuspended in 25% acetonitrile/0.1% trifluoroacetic acid. LMCs are thenseparated on an ABI PTH C18 reverse phase column (220×2.1 mm) using alinear 10%-90% acetonitrile gradient in 0.1% trifluoroacetic acid (flowrate=300 μl/min). The samples were monitored at 214 nm using a ShimadzuUV monitor.

[0153] Results—A comparison was made between a commercially availablebatch of human serum albumin and a batch of rHA purified according tothe invention. Two main significant A₂₁₄ nm peaks are seen in the sampleof the invention (R_(t)=31.1 and 42.8 mins respectively—see FIG. 2 andTable 9). The peak at 2.15 mins is thought to be due to insoluble orpartially soluble material passing through the column, and the largepeak at 56.5 mins is also present in the trace of a water blank and thusis regarded as an artefact. TABLE 5 Peak Results # Ret Time (min) Area(uV.sec) Height (uV) 1 0.800 3459686 219122 2 1.667 418606 33569 3 2.15077883335 1963630 4 3.000 6293258 122295 5 20.433 297608 14424 6 22.900205822 14601 7 27.567 150851 10835 8 31.117 2213883 170938 9 37.983164710 15088 10 39.267 347946 29879 11 41.750 107515 8402 12 42.7832303024 192911 13 43.217 139744 14141 14 43.457 254521 23979 15 50.467152805 13226 16 50.950 162364 12577 17 56.533 5753796 83674

[0154] The commercially available HSA, on the other hand, has many morepeaks (see FIG. 3 and Table 6). TABLE 6 Peak Results # Ret Time (min)Area (uV.sec) Height (uV) 1 0.350 244385 23957 2 0.633 607880 45310 30.783 3239730 243477 4 0.983 1072033 158146 5 2.233 76773569 2038028 62.933 6634089 182363 7 3.733 2812688 95459 8 12.483 818540 20185 912.650 218748 22750 10 14.150 5423715 98336 11 16.333 423403 17460 1216.633 688525 24538 13 17.550 2301309 84781 14 18.033 1145045 47806 1519.750 672721 21562 16 20.233 87799 9760 17 20.700 272171 13003 1821.100 862146 55792 19 21.967 166471 8928 20 22.883 1381445 97660 2123.583 1112632 89851 22 24.000 4740347 419780 23 24.417 352486 26374 2424.917 171279 14625 25 25.133 99734 11473 26 25.267 133911 10515 2725.667 223556 11854 28 25.967 257295 17351 29 26.600 93906 7957 3026.817 223113 18326 31 27.250 303831 29461 32 27.533 124218 12710 3327.783 5747091 561629 34 28.550 1383761 119772 35 29.033 390986 33455 3629.417 182131 12713 37 29.833 181333 12584 38 30.183 478320 30155 3930.583 1048945 58465 40 31.067 3454425 214489 41 31.983 168275 8663 4232.717 651406 43161 43 33.150 1142221 102588 44 34.017 420756 23883 4535.100 115704 10008 46 37.033 166588 9468 47 38.267 145731 8078 4838.983 781209 54029 49 41.800 86967 8868 50 48.883 95416 8522 Si 50.267174159 16737 52 50.483 176115 15573 53 51.267 158727 13701 54 52.183297278 25795 55 56.533 5846645 85710

[0155] The quality of the albumin of the invention in terms ofnon-covalently bound LMCs is clearly superior to that of clinical HSA.Expressed numerically, the total peak area between 10 mins and 55 minsfor the albumin of the invention was about 6.4 V.sec whereas the totalpeak area between the same two times for commercially available materialwas about 39.7 V.sec.

[0156] A similar analysis was carried out with detection at 280 nm, inwhich case the peak area for albumin purified according to the inventionwas 0.56 V.sec, whereas that for HSA was 14.9 V.sec.

[0157] Analysis of fluorescent low molecular weight contaminants(excitation at 280 nm, detection at 350 nm) again revealed a total peakarea for albumin purified by the process of the invention of less than10% of that for HSA.

[0158] Capillary Zone Electrophoresis of rHA and HSA

[0159] Capillary electrophoresis (CE) is used as an alternative tostandard SDS-PAGE in order to qualitatively compare purified rHA of theinvention and commercially available HSA. CE is a high resolvingelectrophoretic technique and is capable of separating sub-populationsof the same protein when only minor differences are to be found.

[0160] Method—Samples of HSA (Armour) and rHA purified according to theinvention were separated in 20 mM PO₄/B₄O₇ buffer, pH=7.4 at 20KeV and30EC were electrophoresed on an ABI 270 CE. The rHA of the inventiongave a single peak on the electrophoretogram indicative of itshomogeneity. In contrast, other peaks were observed in the commerciallyavailable HSA samples. These peaks are believed to be indicative of thepresence of albumin molecules with, for example, blocked free thiolgroups or amino terminal degradation.

[0161] Analysis of C-terminus

[0162] An important aspect of the quality control of recombinantproteins is the confirmation and stability of the pre-determined primarystructure.

[0163] Materials and Methods

[0164] Tryptic Digestion: HSA (from a commercial source—one samplestored at −20EC and one stored at 30EC for 12 weeks), rHA purifiedaccording to the invention (stored at 4EC and 30EC for 6 months) and aDes-Leu rHA (a truncated form of rHA minus the C-terminal leucine) (1 mgeach) were reduced with 5 mM dithiothreitol (Calbiochem) for 120 min37EC, then alkylated with 10 mM iodoacetamide (Sigma) for 90 mins at37EC in 6M guanidine HCl in 0.5M Tris HCl pH 8.0.

[0165] The samples were then diluted 1 in 3 with H₂O and digested withtrypsin for 48 hours at 37EC (TPCK treated trypsin from Sigma, 3×10 μlaliquots of 1 mg/ml solution added over 48 hours).

[0166] Peptide Mapping: Tryptic digests were mapped on reverse phase(RP) HPLC on a Gilson HPLC system using a 25 cm Pharmacia SuperPac Pep-Scolumn (5 μm C₂/C₁₈). The eluents used were A, 0.1% (v/v) TFA (ABI) inwater; B, 0.09% (v/v) TFA in 70% (v/v) acetonitrile (FisonsScientific)—linear gradient over 60 min, 0.5 ml/min. UV detection at 214nm and 280 nm.

[0167] N-terminal Sequencing: Performed on an ABI 477A proteinsequencer.

[0168] Fast Atom Bombardment—Mass Spectrometry: FAB-MS was performed ona VG Autospec by M-Scan Limited, Ascot, UK.

[0169] Peptide Synthesis: The full length C-terminal tryptic peptideLVAASQAALGL (mass 1012) was synthesised by ABI, Warrington, UK; and thetruncated version LVAASQAALG (mass 899) was synthesised by theDepartment of Biochemistry, University of Nottingham, Nottingham, UK.

[0170] Results

[0171] The full length C-terminal tryptic peptide (mass 1012) was shown,using the synthetic marker peptide, to elute at 37.5 minutes on RP-HPLC.This peak was collected and identified by N-terminal Sequencing andFAB-MS from HSA and rHA.

[0172] Removal of the C-terminal leucine results in a truncatedC-terminal peptide (mass 899) which was shown to elute at 28.5 minutes,confirmed using the synthetic marker peptide. This peak was isolatedfrom the tryptic digest of Des-Leu rHA and identified by N-terminalSequencing and FAB-MS. Two other peptides were shown to be present inthis 28.5 minute peak, AWAVAR (mass 673) and DLGEENFK (mass 950).

[0173] The 28.5 minute peak was collected off RP-HPLC from the trypticdigests of HSA, HSA stored at 30EC for 12 weeks, Des-Leu rHA, rHA of theinvention stored at 4EC for 6 months and rHA of the invention stored at30EC for 6 months.

[0174] The peak from each digest was subsequently analysed by N-terminalSequencing and FAB-MS along with the synthetic marker peptides. TABLE 7Peptides present in 28.5 minute peak by N-terminal Sequencing. SAMPLESEQUENCE Des-Leu rHA LVAASQAALG AWAVAR DLGEENFK HSA standard AWAVARDLGEENFK + about 5% LVAASQAALG HSA 30EC 12 weeks AWAVAR DLGEENFK rHA 4EC6 months AWAVAR DLGEENFK rHA 30EC 6 months AWAVAR DLGEENFK

[0175] By FAB-MS, the main signals ((M+H)⁺ molecular ions) present inthe 28.5 minute peak were as shown in Table 8. TABLE 8 (M + H)⁺ Ions in28.5 min Peak. Mixture of Synthetic Full Length and 1013- LVAASQAALGLTruncated C-terminal Peptides 900-LVAASQAALG Des-Leu rHA 673-AWAVAR900-LVAASQAALG 951-DLGEENFK 1028-? 1140-? HSA Standard 673-AWAVAR900-LVAASQAALG 951-DLGEENFK 1028-? 1140-? rHA 30EC 6 months 673-AWAVAR900-LVAASQAALG 1028-? 1140-? 951-No signal

[0176] The signals at 1028 and 1140 may be fragment ions; they were notpeptides that could be detected by sequence analysis.

[0177] Conclusion

[0178] The Des-Leu C-terminal tryptic peptide was detected in commercialHSA at approximately 5-10% n (not quantitative), but could not bedetected in the rHA of the invention, even after 6 months at 30EC. TheDes-Leu peptide could not be detected in the HSA 12 weeks at 30EC, andthe peak for the full length C-terminal peptide at 37.5 minutes (thoughnot isolated) was very diminished compared to the other samples,indicating that perhaps this has undergone further C-terminaldegradation.

[0179] These results indicate that the rHA, purified in accordance withthe invention, has a stable and full length carboxy-terminus, whereasHSA previously available from commercial sources appears to beheterogeneous by comparison.

[0180] Colorimetric Assay for Free Thiols in Purified Human Albumin

[0181] Introduction—Ellmann's Reagent, 5,5N-dithiobis-(2-nitrobenzoate)(DTNB), is a specific and sensitive means of detecting free thiol groupssuch as Cys-SH. The reaction can be followed by monitoring absorbance at412 nm, which value can be used to calculate free Cys-SH, to levels ofless than one residue per molecule of rHA. The following solutionsreagents are utilised in the assay:

[0182] 5,5N-Dithiobis (2-nitrobenzoic acid) DTNB, Sigma Product NoD8130.

[0183] TRIS PRE-SET pH crystals pH8.0, Sigma Product No T4753.

[0184] EDTA, disodium, Sigma Product No ED2SS.

[0185] Sodium dihydrogen phosphate dihydrate, Analar grade.

[0186] Disodium hydrogen phosphate dihydrate, Analar grade.

[0187] Buffer 1: 0.1M (12.1 g) Tris-HCl; 0.01M (3.72 g) EDTA Na₂.2H₂O,pH8.0. PRE-SET pH crystals. Dissolve in 500 ml water and make up to 1litre exact volume. Stable for one month at room temperature.

[0188] Buffer 2: 0.05M Sodium phosphate pH7.0, Na₂HPO₄.2H₂O(5.45 g),3.04 g NaH₂PO₄.2H₂O. Dissolve in 500 ml water, and make up to 1 litreexact volume. Stable for 1 month at room temperature.

[0189] Reagent: 0.01M (39.4 mg) DTNB in phosphate buffer. Dissolve in 10ml buffer 2. Prepare fresh each day.

[0190] Sample: Dilute albumin to about 10.3 μM in buffer 1 (0.66 mg/ml).

[0191] Procedure

[0192] 1) Set spectrophotometer cell holder thermostat to 25EC. 2) Place1.25 ml of sample in one cuvette and 1.25 ml of buffer 1 in another 10mm reduced volume cuvette in the sample and reference positionsrespectively. 3) Zero instrument at 412 nm. Set absorbance to 0.1 AUFull Scale. 4) Add 50 μl DTNB reagent to the reference cuvette, and mixbriefly using a cleaned plastic stirrer. 5) Add 50 μl DTNB reagent tothe sample cuvette, and mix as above. 6) Immediately start acquiringdata (or start chart recorder, and follow reaction for up to 10 mins).7) Repeat for each sample, to obtain values in triplicate. 8)Extrapolate back from the steady absorbance decay to zero time, and readoff the absorbance at 412 nm (δA₄₁₂) (FIG. 1). 9) Calculate thesulphydryl content using the molar extinction coefficient

₄₁₂=13.9 cm²mM⁻¹.

[0193] Results

[0194] A number of commercial HSA samples were assayed for free thiolcontent, the results are summarised below: Free Thiol HSA (mole SH/moleHSA) 1 0.29 2 0.22 3 0.35 4 0.05 5 0.08 6 0.46 7 0.36

[0195] These values are significantly lower than the value for albuminprepared according to the example above which is routinely assayed at0.85-0.9 mole SH/mole rHA.

[0196] The Determination of Metal Ion Contamination in Human Albumin byGraphite Furnace Spectroscopy

[0197] Standards and samples are atomised from a pyrocoated graphitetube. The atomic absorption of the sample is detected using thefollowing conditions:— Atomisation Metal Wavelength temperature ion nmEC Zn 213.9 1800 Cu 327.4 2300 Fe 248.8 2400 Al 309.8 2500 Mn 279.8 2200

[0198] Aluminium was measured using a Perkin Elmer M2100 atomicabsorption spectrophotometer, a Perkin Elmer HGA-700 graphite furnace, aPerkin Elmer AS-70 Autosampler with sample cups and an aluminium hollowcathode lamp. The reagents were AR grade magnesium nitrate, an aluminiumstandard solution (1000 ppm) and AR grade concentrated nitric acid. A1.00% w/v magnesium nitrate solution was made up with Milli-Q water. 15μl of aluminium standard solution was pipetted into the autosampler anddiluted to 1500 μl with 0.20% nitric acid solution. The procedure isrepeated with 15 μl of the solution obtained and then with 150 μL of thesolution subsequently obtained, to give a 10 ppb (μg/L) aluminiumsolution.

[0199] An albumin sample is diluted with 0.20% nitric acid solution togive an aluminium concentration within the limits of the calibrationgraph. A 1:2 dilution is usually sufficient.

[0200] Magnesium is measured similarly, using a Perkin Elmer AS-51 flameautosampler and a magnesium hollow cathode lamp. A Magnesium Standardsolution of 1000 ppm is diluted with Milli-Q water to give 0.1, 0.2, 0.5and 1.0 ppm standard solutions. The atomic absorption of the sample isdetected at 285.2 nm.

[0201] Copper, iron, manganese and zinc are measured in the same way asaluminium except that, for zinc, a 1.0 ppb (μg/l) standard solution isused instead of a 10 ppb solution. The concentration of metal ions wasdetermined in ng/L and then related to the concentration of albumin (ngmetal ion/g albumin). These data are presented in Table 9. TABLE 9Contamination Profiles of Albumin produced according to the inventionConcentration in ng/g albumin Chemical Batch A Batch B Batch C Aluminium— 85 — Copper 3720 9080 1780 Iron 460 810 440 Magnesium 1200 850 800Zinc 4510 1490 1790 Manganese 20 191 16 Chemical Batch D Batch E Batch FBatch G Aluminium — — — — Copper 660 2690 440 530 Iron 930 380 2720 1880Magnesium — — — — Zinc 1580 680 3520 2130 Manganese 42 14 58 27 ChemicalBatch H Batch I Batch J Batch K Aluminium 9 22 86 96 Copper 520 590 99208820 Iron 1010 670 1030 100 Magnesium 600 <400 2000 2000 Zinc 1740 10404280 3520 Manganese 35 20 46 60

[0202] All results are expressed as total metal ion concentration.

[0203] Table 10 shows the corresponding levels of metal ions incommercial HSA. TABLE 10 Concentrations in ng metal/g of albumin SourceA Source B Source C Source D Chemical (UK) (UK) (Japan) (Japan)Aluminium 790 970 915 420 Copper 2020 4510 23840 580 Iron 41220 1520023550 15240 Magnesium 4500 500 15000 54000 Zinc 7230 1650 930 4580Manganese 940 190 135 240 Source E Source F Source G Chemical (UK) (USA)(France) Aluminium 350 3190 155 Copper 4830 1180 7910 Iron 7910 259201850 Magnesium 1500 500 500 Zinc 1520 3940 2130 Manganese 160 65 80

[0204] It can be seen that the average level of aluminium in the productof the invention was about 60 ng/g whereas the commercial sources had155-3190 ng/g. Likewise, the product of the invention had an average ofabout 948 ng/g iron (compare 1850-41,200 ng/g in prior art material), anaverage of 2,990 ng/g of copper (compare 580-23,840 ng/g in prior artmaterial), an average of 1,120 ng/g of magnesium (compare 500-54,000ng/g in prior art material), an average of 2,390 ng/g of zinc (compare930-7,230 ng/g in prior art material, and an average of 48 ng/gmanganese (compare 65 to 940 ng/g in prior art material).

[0205] Analysis of Medium and Long Chain Fatty Acids

[0206] The fatty acids profiles of albumin according to the inventionand commercially available HSA were analysed by acidic solventextraction and gas chromatography of the free fatty acids using a C17:0internal standard.

[0207] Equipment: Gas chromatograph (eg Shimadzu GC 9A) with flameionisation detector; Autoinjector (eg Shimadzu AOC 14);Integrator/Printer (eg Shimadzu CR4A); HP-FFA 30×0.53 mm, 1.0 μm phasecolumn (Hewlett Packard Ltd); Megabore Installation kit (J & WScientific 220-1150 for GC 9A) with direct injection liner.

[0208] Reagents: Water (Milli-Q); Dichloromethane Super Purity Solvent(Romil Chemicals, Loughborough, Leics.); Sodium Acetate TrihydrateAnalar (BDH Ltd, Poole); Acetic Acid Glacial Analar (BDH Ltd, Poole);Human Serum Albumin Solution (Zenalb™20, Bio Products Laboratory,Elstree, Herts.); Sodium Sulphate Anhydrous (Analytical Reagent);standard fatty acids from Sigma.

[0209] Solutions:

[0210] 0.5M Sodium Acetate Buffer pH 4.5: Sodium Acetate 6.13 g andAcetic Acid 3.30 g per 100 ml.

[0211] Free Fatty Acid standard mixtures. Weigh 5 mg of each fatty acidinto separate glass vials. Dissolve each fatty acid in 1 mlDichloromethane and transfer to three 12 ml Pyrex culture tubesrespectively for short chain (C6-C14), medium chain (C16-C18) and longchain (C20-C22:1) fatty acids. Dry down mixture under a stream ofnitrogen and dissolve in 1 ml Dichloromethane. Transfer 50 μl aliquotsof mixture into labelled glass vials, dry under nitrogen, cap and storeat −20EC.

[0212] Internal Standard Solution 1 mg/ml Heptadecanoic Acid (25.0 mgHeptadecanoic Acid/25 ml Dichloromethane).

[0213] Procedure

[0214] 1. Add 50 μl Internal Standard Solution to 6 labelled 40 ml Pyrextubes.

[0215] 2. For 5% rHA add 5 ml sample. For 25% rHA use 1 ml sample and 4ml water. Include a blank (5 ml water) and serum albumin sample (1.25 mlZenalb™20 and 3.75 ml water). Prepare all samples in duplicate.

[0216] 3. Add 2.5 ml Sodium Acetate Buffer, then 10 ml Dichloromethaneto all tubes.

[0217] 4. Place the capped tubes on a mechanical roller for 2 hours atroom temperature.

[0218] 5. Centrifuge all tubes for 5 min at 3,000 rpm in a SorvallRT6000B centrifuge at 20EC.

[0219] 6. Remove the upper aqueous phase, then working from the bottomof the tube carefully transfer the lower Dichloromethane phase into alabelled 12 ml Pyrex tube. Protein globules may hinder the removal ofall the Dichloromethane phase. If this occurs add a spatula full ofAnhydrous Sodium Sulphate, cap and shake.

[0220] 7. Dry Dichloromethane phase under a stream of nitrogen and storeunder nitrogen at −20EC until analysis.

[0221] 8. Install the capillary column and set the gas chromatograph tothe following conditions according to the manufacturer's instructions:—

[0222] Detector: Flame ionisation; Carrier Gas: Nitrogen at 30 ml min⁻¹;Injection Volume: 0.5 μl; Column initial temperature: 70EC; Hold: 1.5min; Gradient 1: 20EC min⁻¹ to 150EC; Gradient 2: 4EC min⁻¹ to 240EC;Hold: 7 min; Detector Temperature: 280EC; Setting Specific to ShimadzuGC9A are: Detector Range: 10E; Hydrogen Pressure: 0.5 kg/cm²; AirPressure: 0.5 kg/cm²; Stop Time: 50 min.

[0223] 9. Set up the integrator to collect data from the gaschromatograph according to the manufacturer's instructions.

[0224] 10. Raise oven temperature to 245EC and leave until a steadybaseline is achieved.

[0225] 11. Lower oven temperature to 70EC and allow to equilibrate.

[0226] 12. Thaw an aliquot of the Long, Medium and Short Chain FattyAcid standards. Dissolve the Long Chain Fatty Acids in 1 mlDichloromethane. Transfer the solution to the Medium Chain Fatty Acidsand dissolve. Repeat for the Short Fatty Acids.

[0227] 13. Inject the standard mixture to determine fatty acid retentiontimes. The chromatogram produced should have very little peak tailingand have a smooth slowly rising baseline with the correct number of wellresolved peaks. Caproic Acid (C6:0) should elute with a retention timeof approx. 6 min and Erucic Acid (C22:1) with a retention time ofapprox. 33 min. Identify all peaks by comparison with example standardchromatogram.

[0228] 14. Inject samples and collect data.

[0229] Calculations

[0230] 1. Identify the internal standard peak from the blank samples.This will be the major peak with a retention time of approximately 23.5min.

[0231] 2. Calculate the Peak Area Ratios for all integrated peaks in allsamples using the following formula.${{Peak}\quad {Area}\quad {Ratio}} = \frac{{Peak}\quad {Area}}{{{Internal}\quad {Standard}\quad {Peak}\quad {Area}}\quad}$

[0232] 3. Identify fatty acid peaks in rHA and HSA samples based onretention time by comparison with standards.

[0233] 4. Convert all Peak Area Ratios to approximate concentrations(μg/g albumin) for both rHA and HSA samples using the following factor:—

Concentration (μg/g)=Peak Area Ratio×200

[0234] 5. For peaks identified as fatty acids convert Concentration fromμg/g albumin to mole/mole albumin using the fatty acid's molecularweight and the following formula:${{Concentration}\left( {{mole}\text{/}{mole}} \right)} = \frac{{{Concentration}\left( {\mu \quad g\text{/}g} \right)} \times 0.0665}{{{Fatty}\quad {Acid}\quad {Molecular}\quad {Weight}}\quad}$

[0235] Example results are presented for a batch of albumin preparedaccording to Example 2 (FIG. 4) and commercial HSA (FIG. 5). No abnormalfatty acids have been detected in the former by this method although theprofiles for the two proteins showed significant differences. Asexpected, both showed large amounts of the added stabiliser, octanoate(C8:0). Apart from this, commercial HSA was characterised bypredominantly C16:0, C16:1, C18:0, C18:1 and C18:2 whilst the albumin ofthe invention contained mainly C10:0, C12:0, C6:1 and occasionallyC14:0. Further experiments showed that the levels of C10:0 and C12:0 inrHA final product correlated with the levels of these contaminants inthe octanoate used for the latter stages of the purification process.

[0236] Data for the rHA produced according to Example 7 are as follows:TABLE 11 Comparison of the fatty acid composition of rHA purifiedaccording to the process of the invention and commercial HSA. Fatty acidcontent (mol/mol protein) Fatty acid rHA HSA C10:0 0.100 0.005 C12:00.020 0.011 C14:0 0.005 0.017 C16:0 0.013 0.152 C16:1 0.064 0.023 C18:00.002 0.024 C18:1 0.012 0.145 C18:2 ND 0.089 C18:3 ND 0.006 C20:0 ND0.001 C20:1 ND 0.001 C20:2 ND ND C20:4 ND 0.006 TOTAL 0.216 0.480

[0237] Preferably, the total level of C18 fatty acids does not exceed1.0% (mole/mole) of the level of octanoate, and preferably does notexceed 0.5% of that level. Moreover, in the albumin of the invention,the level of C18:2, C18:3 and C20 fatty acids is generally undetectable.In commercial HSA, there may typically be about 0.4 moles C10 to C20fatty acids per mole of albumin. In the product of the invention, thereis typically no detectable C20 fatty acids and only about 0.01 to 0.02moles C18 fatty acids per mole of albumin.

[0238] Analysis of Colour—The absorbance of a 5% (w/v) solution of thefinal product in a 1 cm cuvette was measured at 350 nm, 403 nm and 500nm and calculated in terms of absorbances per gram of albumin/litre percm pathlength (ie A L.g⁻¹.cm⁻¹). The albumin of the invention has thefollowing values: Wavelength Mean absorbance (n = 10 batches) (nm)(L.g⁻¹.cm⁻¹) 350 4.74 × 10⁻³ 403 2.12 × 10⁻³ 500 0.58 × 10⁻³

[0239] Generally the albumin of the invention does not exceed respectiveabsorbances of 6.0×10⁻³, 2.5×10⁻³ and 0.75×10⁻³ at the said threewavelengths.

[0240] Assays of number of commercially available HSA preparationsrevealed higher absorbances at these wavelengths (see Table 12). TABLE12 Absorbance (L.g⁻¹.cm⁻¹) of prior art HSA preparations SAMPLE A₃₅₀A₄₀₃ A₅₀₀ 1 9.95 4.10 0.8 2 9.25 5.36 1.1 3 7.40 3.26 0.6 4 7.20 3.600.6 5 8.68 4.08 0.8 6 11.45 6.26 1.2 7 7.20 3.70 0.8 8 6.82 4.78 1.8

[0241] SDS reducing polyacrylamide gel electrophoresis—This assay isperformed to show that rHA consists of a single polypeptide chain whichwhen treated with a reducing agent (β-mercaptoehanol) migrates as asingle band (monomer) on SDS reducing polyacrylamide electrophoresis(PAGE).

[0242] Samples of albumin were boiled in SDS reducing buffer (20 mMTris-HCl pH 8.0 containing 2 mM EDTA, 5% (w/v) SDS and 10% (v/v)β-mercaptoethanol with the albumin at 1 mg/ml, and then separated on SDShomogeneous (12.5%) Phastgels (Pharmacia), using a loading of 1 μl ofthe solution. Protein bands were detected by Coomassie Blue R250staining, scanned on a Shimadzu CS9000 densitometer. Separation ofalbumin showed a single band of Coomassie staining which is indicativethat the proportion of albumin present as a monomer is at least 99.9%.

[0243] Gel Permeation High Pressure Liquid Chromatography

[0244] 25 μl of a 10 mg/ml solution of the albumin in the eluate fromthe anion exchange matrix in the main embodiment of the process of theinvention (ie where the anion exchange step is the final step beforeultrafiltration and formulation) is injected onto a TSK3000SWXL columnon a Shimadzu LC6A HPLC. The product was found to be at least 99.9%monomeric.

[0245] 25 μl of a second 10 mg/ml solution of albumin purified inaccordance with the invention which had been formulated to 25% w/v wasassayed in the same manner and found to contain less than 0.1% polymericalbumin. This result indicates that the formulation as described hereinhas no effect on the polymer/aggregate content of the purified albumin.

[0246] Two Dimensional Gel Electrophoresis

[0247] 2 μg rHA of albumin prepared by the process of the invention wassubject to two-dimensional electrophoresis using a MilliporeInvestigator system. The separation in the first dimension was a pH 3-10isoelectric focusing gel and was followed by a 10% polyacrylamide/SDSgel in the second dimension. On staining of the gel with Coomassie Blue,only one spot was visible, indicating the presence of only one proteinspecies.

[0248] Electrospray Mass Spectrometry

[0249] Electrospray mass spectrometry (ESMS) was performed using a VGQuattro electrospray mass spectrometer, calibrated with horse heartmyoglobin (16951 Da, obtained from Sigma) over m/z range 950-1750 Da/e.Samples of commercially available HSA and samples of rHA purifiedaccording to the invention were desalted prior to analysis by reversephase HPLC using an acetonitrile gradient containing trifluoroaceticacid. FIGS. 6a and b show the spectra for albumin of the invention andprior art HSA, respectively. The latter shows peaks representing blockedfree thiol and N-terminal degradation.

[0250] The albumin of the invention can be seen to be substantiallyhomogeneous in this assay, in other words it shows a single definedpeak, occurring at a mass of about 66441 Da.

[0251] Long Term Stability

[0252] Over two years, no degradation of the albumin is detectable byelectrophoretic methods, which shows that no protease activity ispresent.

1 4 1 11 PRT Homo sapiens 1 Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu1 5 10 2 10 PRT Homo sapiens 2 Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 15 10 3 6 PRT Homo sapiens 3 Ala Trp Ala Val Ala Arg 1 5 4 8 PRT Homosapiens 4 Asp Leu Gly Glu Glu Asn Phe Lys 1 5

1. A process for purifying albumin, the process comprising the steps ofapplying a relatively impure albumin solution to a chromatographicmaterial for which the albumin has no specific affinity such thatalbumin binds to the material, and eluting the bound albumin from thematerial by applying a solution of a compound having a specific affinityfor albumin.
 2. A process according to claim 1 in which thechromatographic material is a cation exchanger resin.
 3. A processaccording to claim 2 wherein the compound is a fatty acid salt such asoctanoate.
 4. A process for purifying albumin according to claim 2, theprocess further comprising anion exchange chromatography in which thealbumin is bound to an anion exchange material.
 5. A process accordingto claim 4 in which albumin eluted from the cation exchange material issubsequently treated by one or more of affinity chromatography,ultrafiltration and gel permeation before being subjected to the saidanion exchange chromatography.
 6. A process according to claim 4 inwhich albumin eluted from the cation exchange material is applied to thesaid anion exchange material without any intervening treatment otherthan dilution.
 7. A process for purifying albumin according to claim 1,comprising the steps of: (a) passing an albumin solution through acation exchange matrix under conditions such that the albumin will bindto the matrix; (b) eluting from said matrix an albumin-containing cationexchange eluate; (c) passing said eluate through an affinity matrixcomprising an albumin-binding compound; (d) eluting from said matrix analbumin-containing affinity matrix eluate; (e) passing said eluatethrough a gel permeation matrix to obtain a fraction enriched inalbumin; (f) passing said albumin-enriched fraction through an anionexchange matrix under conditions such that albumin will bind to thematrix; and (g) eluting from said anion exchange matrix a purifiedalbumin-containing product.
 8. A process for purifying albumin accordingto claim 1, comprising the steps of: (a) passing an albumin solutionthrough a cation exchange matrix under conditions such that the albuminwill bind to the matrix; (b) eluting from the matrix analbumin-containing cation exchange eluate; (c) passing the cationexchange eluate through an anion exchange matrix under conditions suchthat the albumin will bind to the matrix; (d) eluting from the anionexchange matrix an albumin-containing anion exchange eluate; (e) passingthe anion exchange eluate through an affinity matrix comprising analbumin-binding compound; (f) eluting from the affinity matrix analbumin-containing affinity matrix eluate; (g) passing the affinitymatrix eluate through a gel permeation matrix to obtain a fractionenriched in albumin.
 9. A process according to claim 6 wherein thealbumin is eluted in the cation exchange step using a buffer containinga compound having a specific affinity for albumin.
 10. A processaccording to claim 9 wherein the compound is an octanoate salt.
 11. Aprocess according to claim 9 wherein the albumin in the cation exchangestep is washed with a high salt solution before being eluted.
 12. Aprocess according to claim 4 wherein the albumin is eluted from theanion exchanger with a buffer containing 50-200 mM boric acid salt. 13.A process according to claim 1 wherein albumin obtained thereby is then,with or without intervening process steps, subjected to chromatographyon a resin containing an immobilized compound which will selectivelybind glycoconjugates and saccharides.
 14. A process according to claim13 wherein the compound is aminophenylboronic acid (PBA).
 15. A processaccording to claim 5 wherein the affinity chromatography uses a resincomprising an immobilized albumin-specific dye.
 16. A process accordingto claim 15 wherein the dye is a Cibacron Blue type of dye.
 17. Aprocess according to claim 16 wherein the dye is immobilised on theresin via a spacer.
 18. A process according to claim 17 wherein thespacer is an α,ω-diamino-(C₁₋₆-straight chain alkyl) group.
 19. Aprocess according to claim 2 wherein, prior to the cation exchange step,the albumin solution is conditioned by adding octanoate thereto to afinal concentration of from about 1-10 mM and adjusting the pH to about4.0-5.0.
 20. A process according to claim 1 wherein the finalalbumin-containing solution obtained thereby is then ultrafilteredthrough an ultrafiltration membrane to obtain an ultrafiltrationretentate having an albumin concentration of at least about 80 g albuminper litre and the ultrafiltration retentate is diafiltered against atleast 5 retentate equivalents of water.
 21. A process according to claim1 wherein the initial albumin solution is a yeast culture mediumobtained by culturing yeast transformed with an albumin-encodingnucleotide sequence in a fermentation medium, whereby said yeastexpresses and secretes albumin.